Purified Pyrroloquinolinyl-Pyrrolidine-2,5-Dione Compositions And Methods For Preparing And Using Same

ABSTRACT

The present invention relates to a form 1 and form 2 polymorph of (−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin- 1 -yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione. The present invention also relates to (−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin- 1 -yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione compounds having a chiral purity greater than 99%, and methods of preparation of these compounds. The present invention also relates to pharmaceutical compositions comprising these (−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione compounds. The present invention provides methods of treating a cell proliferative disorder, such as a cancer, by administering to a subject in need thereof a therapeutically effective amount of a composition comprising (−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione having a chiral purity greater than 99% or a form 1 and form 2 polymorph of (−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.Provisional Application No. 61/289,563, filed Dec. 23, 2009. Thisapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States,exceeded only by heart disease. (Cancer Facts and Figures. 2004,American Cancer Society, Inc.) Despite recent advances in cancerdiagnosis and treatment, surgery and radiotherapy may be curative if acancer is found early, but current drug therapies for metastatic diseaseare mostly palliative and seldom offer a long-term cure. Even with newchemotherapies entering the market, the need continues for new drugseffective in monotherapy or in combination with existing agents as firstline therapy, and as second and third line therapies in treatment ofresistant tumors.

Cancer cells are by definition heterogeneous. For example, within asingle tissue or cell type, multiple mutational ‘mechanisms’ may lead tothe development of cancer. As such, heterogeneity frequently existsbetween cancer cells taken from tumors of the same tissue and same typethat have originated in different individuals. Frequently observedmutational ‘mechanisms’ associated with some cancers may differ betweenone tissue type and another (e.g., frequently observed mutational‘mechanisms’ leading to colon cancer may differ from frequently observed‘mechanisms’ leading to leukemias). It is therefore often difficult topredict whether a particular cancer will respond to a particularchemotherapeutic agent. (Cancer Medicine, 5th Edition, Bast et al. eds.,B.C. Decker Inc., Hamilton, Ontario)

Components of cellular signal transduction pathways that regulate thegrowth and differentiation of normal cells can, when dysregulated, leadto the development of cellular proliferative disorders and cancer.Mutations in cellular signaling proteins may cause such proteins tobecome expressed or activated at inappropriate levels or atinappropriate times during the cell cycle, which in turn may lead touncontrolled cellular growth or changes in cell-cell attachmentproperties. For example, dysregulation of receptor tyrosine kinases bymutation, gene rearrangement, gene amplification, and overexpression ofboth receptor and ligand has been implicated in the development andprogression of human cancers.

The c-Met receptor tyrosine kinase is the only known high-affinityreceptor for hepatocyte growth factor (HGF), also known as scatterfactor. Binding of HGF to the c-Met extracellular ligand-binding domainresults in receptor multimerization and phosphorylation of multipletyrosine residues in the intracellular portion of c-Met. Activation ofc-Met results in the binding and phosphorylation of adaptor proteinssuch as Gab-1, Grb-2, Shc, and c-Cbl, and subsequent activation ofsignal transducers such as PI3K, PLC-γ, STATs, ERK1 and 2 and FAK. c-Metand HGF are expressed in numerous tissues, and their expression isnormally confined predominantly to cells of epithelial and mesenchymalorigin, respectively. c-Met and HGF are dysregulated in human cancersand may contribute to dysregulation of cell growth, tumor celldissemination, and tumor invasion during disease progression andmetastasis (See, e.g., Journal of Clinical Investigation 109: 863-867(2002) and Cancer Cell pp 5-6 Jul. 2004). c-Met and HGF are highlyexpressed relative to surrounding tissue in numerous cancers, and theirexpression correlates with poor prognosis and lack of response tostandard clinical treatments. (See, e.g., Journal of CellularBiochemistry 86: 665-677 (2002); Int. J. Cancer (Pred. Oncol.) 74:301-309 (1997); Clinical Cancer Research 9: 1480-1488 (2003); and CancerResearch 62: 589-596 (2002)). Without intending to be bound by theory,c-Met and HGF may protect tumors against cell death induced byDNA-damaging agents and, as such, may contribute to chemoresistance andradioresistance of tumors. Without intending to be limited by anytheory, inhibitors of c-Met may be useful as therapeutic agents in thetreatment of proliferative disorders including breast cancer. (See,e.g., Cancer and Metastasis Reviews 22: 309-325 (2003)). Accordingly,new compounds and methods for modulating these factors and treatingcancer are needed. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention provides a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation. In someembodiments, the polymorph can also be characterized by an X-ray powderdiffraction pattern comprising peaks at approximately 8.2, 10.8, 14.1,15.5, 17.8, 19.9 and 25.6° 2θ using Cu Kα radiation. In otherembodiments, the polymorph can also be characterized by an X-ray powderdiffraction pattern comprising peaks at approximately 8.2, 10.8, 14.1,14.9, 15.5, 17.1, 17.8, 19.4, 19.9, 21.1, 21.9, 23.0, 25.6 and 28.4° 2θusing Cu Kα radiation.

The present invention also provides a form 2 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation. In someembodiments, the polymorph can also be characterized by an X-ray powderdiffraction pattern comprising peaks at approximately 6.5, 9.9, 12.0,16.7, 20.1 and 22.8° 2θ using Cu Kα radiation. In other embodiments, thepolymorph can also be characterized by an X-ray powder diffractionpattern comprising peaks at approximately 6.5, 9.9, 12.0, 13.2, 16.4,16.7, 17.2, 20.1, 20.3, 20.8, 22.8, 23.7, 28.6 and 30.4° 2θ using Cu Kαradiation.

The present invention also provides a(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane and a composition comprising(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane. The composition can comprise greater than 90%, greaterthan 95% or greater than 99%(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane.

The present invention also provides a(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrineor a composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.The composition can comprise greater than 90%, greater than 95% orgreater than 99%(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.In some embodiments, the composition can comprise less than 1%, lessthan 0.5% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine.

The present invention also provides a chirally purified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprising less than 1%, less than 0.7%, less than 0.5% or less than0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The present invention also provides a method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: (a) mixing(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewith (1S,2S)-(+)-pseudoephedrine in a first solvent to form solid(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine;(b) washing the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinesolid formed in step (a) with an aqueous mixture of the first solvent;(c) reacting the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinefrom step (b) with an acid in an organic solvent and isolating theorganic layer of the resultant solution; (d) washing the organic layerfrom step (c); (e) adding a second solvent to the organic layer; (f)concentrating the organic layer until the amount of the second solventin the solution is less than 5%; and (g) crystallizing from the organiclayer in step (f) and drying the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionesolution under vacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Preferably, the produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%, less than 0.7%, less than 0.5% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The first solvent can be a non-aqueous solvent. Preferably, the firstnon-aqueous solvent can be methanol, ethanol, cyclohexylethylamine,acetonitrile, or a mixture thereof. The second solvent can be anon-aqueous solvent. Preferably, the second non-aqueous solvent can bemethanol, ethanol, acetonitrile, or a mixture thereof. In someembodiments, the second solvent is the same as said first solvent. Inother embodiments, the second solvent is different from said firstsolvent. The organic solvent in step (c) can be methyltetrahydrofuran.In some embodiments, the organic layer is washed with a salt solution instep (d). Preferably, the salt solution is a sodium chloride solution.

The method can further include rinsing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal after step (g). In some embodiments, the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal is rinsed with an alcohol. Preferably, the alcohol is selectedfrom ethanol and methanol.

The present invention also provides a method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising (a) mixing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrineand an acid; (b) adding an alcohol to the mixture from (a) to form aslurry; (c) heating and stirring the slurry formed in (b); (d) coolingand isolating(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;(e) washing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneisolated in step (d) with a first solvent; (f) dissolving(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom step (e) in a second solvent to form a solution; (g) adding a thirdsolvent to the solution in (f) and distilling the solution until theamount of said second solvent in the solution is less than 5%; (h)crystallizing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom the solution in (g); (i) optionally, adding a fourth solvent(preferably water) to mature the crystals from (h); (j) isolating thecrystals from (i) by filtration; (k) washing the crystals from (j) witha mixture of the third solvent and fourth solvent; and (l) drying thecrystals from (k) under vacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Preferably, the produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%, less than 0.7%, less than 0.5% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The alcohol can be methanol, ethanol, or a mixture thereof. The firstsolvent can be a non-aqueous solvent. Preferably, the first non-aqueoussolvent can be methanol, ethanol, or a mixture thereof. The secondsolvent can be a non-aqueous solvent. Preferably, the second non-aqueoussolvent is tetrahydrofuran. The third solvent can be a non-aqueoussolvent. Preferably, the third non-aqueous solvent can be methanol,ethanol, or a mixture thereof.

The present invention also provides a method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: (a) dissolving(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein dichloromethane and isolating the(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane; (b) dissolving(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane in a first solvent; (c) distilling the solution of step(b) until the level of dichloromethane in the solution is <0.1% byweight; (d) diluting the solution of step (c) in a second solvent; (e)introducing the solution of step (d) into a multicolumn chromatographysystem containing a packing suitable for chiral separation; (f) poolingthe resultant raffinate obtained from the system in step (e); and (g)crystallizing the raffinate from step (f) and filtering the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor evaporating the raffinate from step (f) to dryness, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Preferably, the produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%, less than 0.7%, less than 0.5% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Preferably, the first non-aqueous solvent can be methanol, ethanol, or amixture thereof. The second solvent can be a non-aqueous solvent.Preferably, the second non-aqueous solvent can be methanol, ethanol,acetonitrile, or a mixture thereof. More preferably, the secondnon-aqueous solvent is a mixture of methanol and acetonitrile.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 sets forth the chemical structures of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 2 sets forth an effect of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneon survival of MDA-MB-231 or Paca-2 cells in vitro.

FIG. 3 sets forth an effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneon survival of MDA-MB-231 cells in vitro.

FIG. 4 sets forth an effect of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneon Protein Kinase C activity in vitro.

FIG. 5, Panel A sets forth inhibition of autophosphorylation of c-Met by(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;Panel B sets forth inhibition of induced c-Met phosphorylation by(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 6 sets forth an effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneto induce apoptosis in cancer cells.

FIG. 7 sets forth an effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneto inhibit metastatic cancer cell invasion.

FIG. 8 sets forth an effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneon breast cancer xenograft model.

FIG. 9 sets forth an effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneon human colon cancer xenograft model (Panel A), human pancreatic cancerxenograft model (Panel B), human prostate cancer xenograft model (PanelC), and human gastric cancer xenograft model (Panel D).

FIG. 10 sets forth cytotoxic sensitivity of multiple cell lines to(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 11 sets forth reduction in the amount of phosphorylated c-Met inhistopathological samples treated with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneby immunohistochemistry (Panel A) or by Western blotting (Panel B).

FIG. 12, Panels A sets forth the XRPD pattern of Form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;Panel B sets forth typical 2θ values of the XRPD pattern of Form 1polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 13, Panel A sets forth the XRPD pattern of Form 2 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;Panel B sets forth typical 2θ values of the XRPD pattern of Form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 14 sets forth graphics comparing the XRPD patterns of Forms 1 and 2polymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 15 Panels A and B set forth graphics showing the IR spectra ofForms 1 and 2 of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 16 sets forth graphics showing the thermal (melting) behavior of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneForms 1 and 2.

FIG. 17, Panels A and B set forth graphics showing the solubility andintrinsic dissolution of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneForms 1 and 2, depicted as Forms B and A, respectively.

DETAILED DESCRIPTION OF THE INVENTIONPyrroloquinolinyl-Pyrrolidine-2,5-Dione Compounds

The present invention provides(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The present invention also provides a composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.The composition can comprise one or more pharmaceutically acceptablecarriers or excipients.

Preferably, the highly purified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneprovided by the instant invention has a chiral purity greater than99.3%, greater than 99.5%, greater than 99.6%, greater than 99.7%,greater than 99.8% or greater than 99.9%. Preferably, the compositionscontaining highly purified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione provided by the instant invention contain lessthan 0.7%, less than 0.5%, less than 0.4%, less than 0.3%, less than0.2% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

All forms of the compounds of the instant invention are contemplated,either in admixture or in pure or substantially pure form, includingcrystalline forms of racemic mixtures and crystalline forms ofindividual isomers. The invention very particularly embraces isolatedoptical isomers having a specified activity. The racemic forms can beresolved by physical methods, such as, for example, separation orcrystallization of diastereomeric derivatives, separation by chiralcolumn chromatography or supercritical fluid chromatography. Theindividual optical isomers can be obtained from the racemates byconventional methods, such as, for example, salt formation with anoptically active acid or base followed by crystallization.

Certain compounds of this invention may exist in tautomeric forms. Allsuch tautomeric forms of the compounds are considered to be within thescope of this invention unless otherwise stated.

In addition, a crystal polymorphism may be present but is not limiting,but any crystal form may be single or a crystal form mixture, or ananhydrous or hydrated crystal form.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectra, melting points,density, crystal shape, optical and electrical properties, stability andsolubility. Crystallization solvent, rate of crystallization, storagetemperature, and other factors may cause one crystal form to dominate.Crystal polymorphs of the compounds can be prepared by crystallizationunder different conditions.

The present invention provides two polymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

A form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation. In someembodiments, the polymorph can also be characterized by an X-ray powderdiffraction pattern comprising peaks at approximately 8.2, 10.8, 14.1,15.5, 17.8, 19.9 and 25.6° 2θ using Cu Kα radiation. In otherembodiments, the polymorph can also be characterized by an X-ray powderdiffraction pattern comprising peaks at approximately 8.2, 10.8, 14.1,14.9, 15.5, 17.1, 17.8, 19.4, 19.9, 21.1, 21.9, 23.0, 25.6 and 28.4° 2θusing Cu Kα radiation.

The present invention also provides a form 2 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation. In someembodiments, the polymorph can also be characterized by an X-ray powderdiffraction pattern comprising peaks at approximately 6.5, 9.9, 12.0,16.7, 20.1 and 22.8° 2θ using Cu Kα radiation. In other embodiments, thepolymorph can also be characterized by an X-ray powder diffractionpattern comprising peaks at approximately 6.5, 9.9, 12.0, 13.2, 16.4,16.7, 17.2, 20.1, 20.3, 20.8, 22.8, 23.7, 28.6 and 30.4° 2θ using Cu Kαradiation.

The present invention also provides a composition comprising the form 1polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation. Thecomposition can comprise one or more pharmaceutically acceptablecarriers or excipients.

The present invention also provides a composition comprising the form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation. Thecomposition can comprise one or more pharmaceutically acceptablecarriers or excipients.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

The present invention also provides a(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.The(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinecan comprise less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine.

The present invention also provides a composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine. The composition can comprise one or morepharmaceutically acceptable carriers or excipients.

Preferably, the highly purified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrineprovided by the instant invention have a diastereomeric purity greaterthan 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%or greater than 99.9%. Preferably, the compositions containing highlypurified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrineprovided by the instant invention contain less than 0.5%, less than0.4%, less than 0.3%, less than 0.2% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate. For example, the solvate may be adichloromethane (DCM) solvate.

The present invention also provides a(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane and a composition comprising(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane. The composition can comprise greater than 90%, greaterthan 95% or greater than 99%(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane. The composition can comprise one or morepharmaceutically acceptable carriers or excipients.

Some compounds of the present invention can exist in a tautomeric formwhich are also intended to be encompassed within the scope of thepresent invention. “Tautomers” refers to compounds whose structuresdiffer markedly in arrangement of atoms, but which exist in easy andrapid equilibrium. It is to be understood that the compounds of theinvention may be depicted as different tautomers. It should also beunderstood that when compounds have tautomeric forms, all tautomericforms are intended to be within the scope of the invention, and thenaming of the compounds does not exclude any tautomeric form.

The compounds, salts and prodrugs of the present invention can exist inseveral tautomeric forms, and such tautomeric forms are included withinthe scope of the present invention. Tautomers exist as mixtures of atautomeric set in solution. In solid form, usually one tautomerpredominates. Even though one tautomer may be described, the presentinvention includes all tautomers of the present compounds

As used herein, the term “salt” is a pharmaceutically acceptable saltand can include acid addition salts including hydrochlorides,hydrobromides, phosphates, sulphates, hydrogen sulphates,alkylsulphonates, arylsulphonates, acetates, benzoates, citrates,maleates, fumarates, succinates, lactates, and tartrates; alkali metalcations such as Na⁺, K⁺, Li⁺, alkali earth metal salts such as Mg²⁺ orCa²⁺, or organic amine salts.

As used herein, the term “metabolite” means a product of metabolism of acompound of the present invention, or a pharmaceutically acceptablesalt, polymorph or solvate thereof, that exhibits a similar activity invivo to said compound of the present invention.

As used herein, the term “mixing” means combining, blending, stirring,shaking, swirling or agitating. The term “stirring” means mixing,shaking, agitating, or swirling. The term “agitating” means mixing,shaking, stirring, or swirling.

The compounds of the present invention can also be prepared as prodrugs,for example pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. The term “prodrug” includes a compound of the presentinvention covalently linked to one or more pro-moieties, such as anamino acid moiety or other water-solubilizing moiety. A compound of thepresent invention may be released from the pro-moiety via hydrolytic,oxidative, and/or enzymatic release mechanisms. In an embodiment, aprodrug composition of the present invention exhibits the added benefitof increased aqueous solubility, improved stability, and improvedpharmacokinetic profiles. The pro-moiety may be selected to obtaindesired prodrug characteristics. For example, the pro-moiety, e.g., anamino acid moiety or other water solubilizing moiety such as phosphatemay be selected based on solubility, stability, bioavailability, and/orin vivo delivery or uptake. The term “prodrug” is also intended toinclude any covalently bonded carriers that release an active parentdrug of the present invention in vivo when such prodrug is administeredto a subject. Prodrugs in the present invention are prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound. Prodrugs include compounds of the presentinvention wherein a hydroxy, amino, sulfhydryl, carboxy, or carbonylgroup is bonded to any group that, may be cleaved in vivo to form a freehydroxyl, free amino, free sulfhydryl, free carboxy or free carbonylgroup, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters groups (e.g. ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g. N-acetyl) N-Mannich bases, Schiff bases and enaminonesof amino functional groups, oximes, acetals, ketals and enol esters ofketone and aldehyde functional groups in compounds of Formula I, and thelike, See Bundegaard, H. “Design of Prodrugs” p 1-92, Elesevier, NewYork-Oxford (1985).

Synthesis of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneCompounds

Standard synthetic methods and procedures for the preparation of organicmolecules and functional group transformations and manipulations,including the use of protective groups, can be obtained from therelevant scientific literature or from standard reference textbooks inthe field. Although not limited to any one or several sources,recognized reference textbooks of organic synthesis include: Smith, M.B.; March, J. March's Advanced Organic Chemistry Reactions, Mechanisms,and Structure, 5^(th) ed.; John Wiley & Sons: New York, 2001; andGreene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis,3rd; John Wiley & Sons: New York, 1999. The following descriptions ofsynthetic methods are designed to illustrate, but not limit, generalprocedures for the preparation of compounds of the invention.

The present invention also provides a method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: (a) mixing(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewith (1S,2S)-(+)-pseudoephedrine in a first solvent to form solid(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine;(b) washing the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinesolid formed in step (a) with an aqueous mixture of the first solvent;(c) reacting the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinefrom step (b) with an acid in an organic solvent and isolating theorganic layer of the resultant solution; (d) washing the organic layerfrom step (c); (e) adding a second solvent to the organic layer; (f)concentrating the organic layer until the amount of the second solventin the solution is less than 5%; and (g) crystallizing from the organiclayer in step (f) and drying the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneunder vacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Preferably, the produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%, less than 0.7%, less than 0.5% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The first solvent can be a non-aqueous solvent. Preferably, the firstnon-aqueous solvent can be methanol, ethanol, acetonitrile, or a mixturethereof. The second solvent can be a non-aqueous solvent. Preferably,the second non-aqueous solvent can be methanol, ethanol, acetonitrile,or a mixture thereof. In some embodiments, the second solvent is thesame as said first solvent. In other embodiments, the second solvent isdifferent from said first solvent. The organic solvent in step (c) canbe methyltetrahydrofuran. In some embodiments, the organic layer iswashed with a salt solution in step (d). Preferably, the salt solutionis a sodium chloride solution.

The method can further include rinsing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal after step (g). In some embodiments, the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal is rinsed with an alcohol. Preferably, the alcohol is selectedfrom ethanol and methanol.

The present invention also provides a method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising (a) mixing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrineand an acid; (b) adding an alcohol to the mixture from (a) to form aslurry; (c) heating and stirring the slurry formed in (b); (d) coolingand isolating(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;(e) washing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneisolated in step (d) with a first solvent; (f) dissolving(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom step (e) in a second solvent to form a solution; (g) adding a thirdsolvent to the solution in (f) and distilling the solution until theamount of said second solvent in the solution is less than 5%; (h)crystallizing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom the solution in (g); (i) optionally, adding a fourth solvent(preferably water) to mature the crystals from (h); (j) isolating thecrystals from (i) by filtration; (k) washing the crystals from (j) witha mixture of the third solvent and fourth solvent; and (l) drying thecrystals from (k) under vacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The alcohol can be methanol, ethanol, or a mixture thereof. The firstsolvent can be a non-aqueous solvent. Preferably, the first non-aqueoussolvent can be methanol, ethanol, or a mixture thereof. The secondsolvent can be a non-aqueous solvent. Preferably, the second non-aqueoussolvent is tetrahydrofuran. The third solvent can be a non-aqueoussolvent. Preferably, the third non-aqueous solvent can be methanol,ethanol, or a mixture thereof. The fourth solvent can be an aqueoussolvent. Preferably, the fourth solvent is water.

The present invention also provides a method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: (a) dissolving(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein dichloromethane and isolating the(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane; (b) dissolving(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane in a first solvent; (c) distilling the solution of step(b) until the level of dichloromethane in the solution is <0.1% byweight; (d) diluting the solution of step (c) in a second solvent; (e)introducing the solution of step (d) into a multicolumn chromatographysystem containing a packing suitable for chiral separation; (f) poolingthe resultant raffinate obtained from the system in step (e); and (g)crystallizing the raffinate from step (f) and filtering the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor evaporating the raffinate from step (f) to dryness, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Preferably, the produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%, less than 0.7%, less than 0.5% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Preferably, the first non-aqueous solvent can be methanol, ethanol, or amixture thereof. The second solvent can be a non-aqueous solvent.Preferably, the second non-aqueous solvent can be methanol, ethanol,acetonitrile, or a mixture thereof. More preferably, the secondnon-aqueous solvent is a mixture of methanol and acetonitrile.

Scheme I provides a summary for the production of the instantcompositions comprising highly purified (>99% chiral purity)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,with minimal amounts (<1%) of the undesired enantiomer,(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,present. Preferably, the compositions comprise(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99.3%, greater than 99.5%, greaterthan 99.6%, greater than 99.7%, greater than 99.8% or greater than99.9%. Preferably, the compositions comprise less than 0.7%, less than0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

As shown in scheme I and described in detail in the following examples,various procedures are utilized to produce and isolate thesecompositions comprising highly chirally pure(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,including but not limited to, multicolumn chromatography resolution,diastereomeric salt resolution or dynamic kinetic resolution.

In step 1 of scheme I, lilolidine (Compound 4), was treated withoxalylchloride to give the acyl chloride in methyl tert-butyl ether(MTBE) reaction solvent. The addition of methanol was sufficient toafford the intermediate ketoester,5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acid methylester (Compound 5). In Step 2 of the reaction, Compound 5 andindole-3-acetamide (Compound 5a) were combined to produce3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione(Compound 6). As described in detail in the following examples, it isnot necessary to isolate intermediate Compound 5 to produce Compound 6.A solvent swap from MTBE into tetrahydrofuran (THF) was necessary priorto conducting Step 2, since both 5 and 5a have very poor solubility inMTBE. Following the addition of Compound 5a to consume Compound 5 in THFin the presence of base, HCl was added to complete the reaction toafford crude Compound 6. Crude Compound 6 was then purified from DCM andheptane to afford Compound 6 of sufficient purity to proceed to the nextstep. A detailed description of this process is shown in Example 1.

Compound 6 was hydrogenated with palladium hydroxide, THF, and potassiumtert-butoxide to produce crude trans racemate,(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 8). As described in the examples, the preparation of Compound8, does not require the isolation of crude cis racemate(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 7). Rather, during the hydrogenation of Compound 6, conditionswere modified to effect the isomerization of the resultant Compound 7 insitu. The resulting process provides a means to control key impuritiesin Compound 8, specifically Compound 6. Specifically, the reaction iscarried out in THF solvent, and the catalyst employed is palladiumhydroxide. Potassium tert-butoxide is also added to the reaction mixtureto promote the isomerization. A detailed description of this process isshown in Example 2.

The most common impurity in Compound 8 is typically Compound 7. SinceCompound 7 is itself chiral, it is particularly difficult to predict theelution of isomers of Compound 7 from Multicolumn Chromatography (MCC)when lower purity (<99%) Compound 8 is utilized. In order to ensure highpurity product, it is, therefore, necessary to increase the purity ofthe racemate, Compound 8, prior to enantiomeric resolution by MCC. Itwas determined that the purity of Compound 8 could be improved throughselective crystallization of solvates from various solvents. One suchsolvate formed from dichloromethane (DCM) yields Compound 8 DCM withvery high chemical purity (>99%). As shown in scheme I, the Compound 8DCM was formed by dissolving the crude Compound 8 in DCM and thenseeding with DCM crystals. After growing the seed bed to a criticalmass, heptane was added to drive the crystallization to completion.After isolation by filtration and removal of bulk residual solvent undervacuum, Compound 8 DCM typically contained 0.8-0.9 moles of DCM withrespect to Compound 8. This solvate crystallization removes the processimpurities present in crude (95-98%) Compound 8 as a result of thehydrogenation and isomerization. A detailed description of this processis shown in Example 3.

The present invention provides the chiral resolution of the twoenantiomers of Compound 8 DCM by MCC, as shown in Scheme I. The MCCenantiomeric separation process provides an advantage over separation byHPLC batch chromatography in that it can provide high chiral purity(>99%) on larger scale (>20 kg) than batch preparation. MCC resolutioncan be carried out in either methanol, a mixture of methanol and ethanol(1:1 vol) or methanol and acetonitrile (9:1 v/v). The chiral stationaryphase (CSP) utilized is either Chiralpak AD or AZ purchased from ChiralTechnologies, Inc. The concentration of the racemate feed solution is inthe range of 20-50 g/L. Preferably, the specified chiral purity for theseparation is >99% of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 10) and containing <1% of(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 9).

Specifically, separations are carried out by preparing a feed solutionof racemic Compound 8 in the chosen mobile phase. This solution isprepared from Compound 8 DCM by initially dissolving the material inmethanol. The chromatographic parameters were estimated prior toproduction, and final optimization was conducted during the initialhours or days of separation of the feed solution. Where initialfractions collected did not meet the chiral purity specification, thematerial was recycled into the feed solution to repeat the resolution.Once the operating parameters to meet specifications had beenestablished, the conditions were applied to separate the entire volumeof feed solution. Throughout the operation, Compound 10 was collected asthe raffinate stream, pooled, and concentrated. The concentratedraffinate was then either concentrated to dryness (<5 kg scale) orconcentrated and seeded to induce crystallization of Compound 10 (>5 kgscale). A detailed description of this process is shown in Example 4.

In addition to MCC resolution, a classical approach for the resolutionof chiral acids or bases through the formation of diastereomeric saltshas been employed. In the case of Compound 10, either acids or basescould be used, since the molecule is amphoteric. Following a detailedscreen of chiral bases, it was shown that the salts formed from theenantiomers of Compound 8 with pseudoephedrine have vastly differentsolubility profiles in some solvents. This relationship was optimized toallow for the resolution of Compound 8 using (1S,2S)-(+)-pseudoephedrineto afford(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine(Compound 10.(1S,2S)-(+)-pseudoephedrine) selectively. A detaileddescription of this process is shown in Example 6. Another processprovided by the instant invention is to racemize the undesiredenantiomer,(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 9), in situ using Dynamic Kinetic Resolution (DKR). A detaileddescription of this process is shown in Example 7.

In both the MCC resolution and classical diastereomeric salt resolutionprocess, the undesired enantiomer, Compound 9, is isolated. Thisundesired enantiomer can be isomerized to obtain racemic Compound 8.This is accomplished by implementing the same procedure for isomerizingthe cis-isomer, Compound 7, to afford crude trans racemate Compound 8.The isomerization is carried out in either methanol or ethanol usingsodium hydroxide as a base. A detailed description of this process isshown in Example 5.

The synthesis methods described in Scheme I are readily reproducible ona large scale, e.g., 10 kg, 20 kg, 30 kg, 40 kg, 50 kg, and 60 kg andhigher providing an overall yield of >45% of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 10), based on indole acetamide as a limiting reagent.Preferably, 1 kg of lilolidine (Compound 4) yields approximately 1 kg of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 10) having a purity >99%.

The present invention also provides a method to prepare polymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.The crude Compound 10 was dissolved in a solvent, for example, THF, byheating the resulting mixture to 50° C. The resulting mixture was polishfiltered, followed by the addition of a second solvent, such asmethanol, and a Form 1 seed crystal of Compound 10.

The use of seed crystals imparts polymorphic control of thecrystallization process. In the absence of seeding, either Form 1 orForm 2 can be spontaneously crystallized in high purity. Thus, toprovide Form 1 or Form 2 seeds, spontaneous crystallization ispermitted; and once formed, the crystalline product can be characterizedusing quantitative analysis by x-ray powder diffraction to determine theresulting crystal form and its polymorphic purity. Following suchcharacterization, this crystalline product can be used as “seedcrystals” or “seeds” to control the polymorph, either Form 1 or Form 2,generated during the subsequent crystallizations as described herein.

The solution was then concentrated azeotropically and atmospherically bydistillation to reduce the volume of the solvent, e.g., THF. Thetemperature of the solution was reduced to 50° C. and stirred for atleast 4 hours. Aliquots were removed to confirm to formation of thedesired polymorph. If required, the polymorph can be redissolved in THF(30% of batch volume), polish filtered, concentrated and seeded toobtain the desired polymorphic outcome. When the desired polymorphicform was obtained, a solution of 50% aqueous methanol was added at 50°C. and the solution was agitated for an additional 2-3 hours. Thesolution was then cooled to ambient temperature and held for at least 2hours to allow crystallization. Upon completion, the crystals wereisolated by filtration, washed with additional 50% aqueous methanol, anddried under vacuum at 65° C. for at least 12 hours. Polymorphic Form 1of Compound 10 was isolated as a red-brown solid.

Form 2 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecan also be prepared with the above method if the Form 1 seed crystal isreplaced by the Form 2 seed crystal.

Methods of Treatment

The present invention provides methods for the treatment of a cellproliferative disorder in a subject in need thereof by administering toa subject in need of such treatment, a therapeutically effective amountof a pharmaceutical composition comprising (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than containing less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients.

The cell proliferative disorder can be cancer or a precancerouscondition. The present invention further provides the use of acomposition of (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients for thepreparation of a medicament useful for the treatment of a cellproliferative disorder.

The present invention also provides methods of protecting against a cellproliferative disorder in a subject in need thereof by administering atherapeutically effective amount of a pharmaceutical compositioncomprising (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than containing less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients, and one or morepharmaceutically acceptable carriers or excipients to a subject in needof such treatment. The cell proliferative disorder can be cancer or aprecancerous condition. The present invention also provides the use of acompound of (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than containing less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients. for thepreparation of a medicament useful for protecting against a cellproliferative disorder.

As used herein, a “subject in need thereof” is a subject having a cellproliferative disorder, or a subject having an increased risk ofdeveloping a cell proliferative disorder relative to the population atlarge. A subject in need thereof can have a precancerous condition.Preferably, a subject in need thereof has cancer. A “subject” includes amammal. The mammal can be e.g., any mammal, e.g., a human, primate,bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or apig. Preferably, the mammal is a human.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue. A cell proliferative disorder includes aprecancer or a precancerous condition. A cell proliferative disorderincludes cancer. Preferably, the methods provided herein are used totreat or alleviate a symptom of cancer. The term “cancer” includes solidtumors, as well as, hematologic tumors and/or malignancies. A “precancercell” or “precancerous cell” is a cell manifesting a cell proliferativedisorder that is a precancer or a precancerous condition. A “cancercell” or “cancerous cell” is a cell manifesting a cell proliferativedisorder that is a cancer. Any reproducible means of measurement may beused to identify cancer cells or precancerous cells. Cancer cells orprecancerous cells can be identified by histological typing or gradingof a tissue sample (e.g., a biopsy sample). Cancer cells or precancerouscells can be identified through the use of appropriate molecularmarkers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, urinarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma,Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular(eye) melanoma, merkel cell carcinoma, mesothelioma malignant,mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer ofthe tongue, multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. Preferably,compositions of the present invention may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent invention or a hematologic cell proliferative disorder of thepresent invention. A hematologic cancer of the present invention caninclude multiple myeloma, lymphoma (including Hodgkin's lymphoma,non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas oflymphocytic and cutaneous origin), leukemia (including childhoodleukemia, hairy-cell leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloidneoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. Preferably, compositions of the present invention may be usedto treat lung cancer or cell proliferative disorders of the lung. Lungcancer can include all forms of cancer of the lung. Lung cancer caninclude malignant lung neoplasms, carcinoma in situ, typical carcinoidtumors, and atypical carcinoid tumors. Lung cancer can include smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),non-squamous non-small cell lung cancer, squamous non-small cell lungcancer, squamous cell carcinoma, non-squamous cell carcinoma,adenocarcinoma, small cell carcinoma, large cell carcinoma,adenosquamous cell carcinoma, and mesothelioma. Lung cancer can include“scar carcinoma,” bronchioalveolar carcinoma, giant cell carcinoma,spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lungcancer can include lung neoplasms having histologic and ultrastructualheterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. Preferably,compositions of the present invention may be used to treat colon canceror cell proliferative disorders of the colon. Colon cancer can includeall forms of cancer of the colon. Colon cancer can include sporadic andhereditary colon cancers. Colon cancer can include malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. Compositions of the present invention may beused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of N0; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer.Preferably, compositions of the present invention may be used to treatbreast cancer. Breast cancer includes all forms of cancer of the breast.Breast cancer can include primary epithelial breast cancers. Breastcancer can include cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. Breast cancer can includecarcinoma of the breast, ductal carcinoma of the breast, lobularcarcinoma of the breast, undifferentiated carcinoma of the breast,cystosarcoma phyllodes of the breast, angiosarcoma of the breast, andprimary lymphoma of the breast. Breast cancer can include Stage I, II,IIIA, IIIB, IIIC and IV breast cancer. Ductal carcinoma of the breastcan include invasive carcinoma, invasive carcinoma in situ withpredominant intraductal component, inflammatory breast cancer, and aductal carcinoma of the breast with a histologic type selected from thegroup consisting of comedo, mucinous (colloid), medullary, medullarywith lymphcytic infiltrate, papillary, scirrhous, and tubular. Lobularcarcinoma of the breast can include invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. Breast cancer can include Paget'sdisease, Paget's disease with intraductal carcinoma, and Paget's diseasewith invasive ductal carcinoma. Breast cancer can include breastneoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

Preferably, a compound of the present invention may be used to treatbreast cancer. A breast cancer that is to be treated can includefamilial breast cancer. A breast cancer that is to be treated caninclude sporadic breast cancer. A breast cancer that is to be treatedcan arise in a male subject. A breast cancer that is to be treated canarise in a female subject. A breast cancer that is to be treated canarise in a premenopausal female subject or a postmenopausal femalesubject. A breast cancer that is to be treated can arise in a subjectequal to or older than 30 years old, or a subject younger than 30 yearsold. A breast cancer that is to be treated has arisen in a subject equalto or older than 50 years old, or a subject younger than 50 years old. Abreast cancer that is to be treated can arise in a subject equal to orolder than 70 years old, or a subject younger than 70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

A compound of the present invention may be used to treat or prevent acell proliferative disorder of the breast, or to treat or prevent breastcancer, in a subject having an increased risk of developing breastcancer relative to the population at large. A subject with an increasedrisk of developing breast cancer relative to the population at large isa female subject with a family history or personal history of breastcancer. A subject with an increased risk of developing breast cancerrelative to the population at large is a female subject having agerm-line or spontaneous mutation in BRCA1 or BRCA2, or both. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female subject with a family history of breastcancer and a germ-line or spontaneous mutation in BRCA1 or BRCA2, orboth. A subject with an increased risk of developing breast cancerrelative to the population at large is a female who is greater than 30years old, greater than 40 years old, greater than 50 years old, greaterthan 60 years old, greater than 70 years old, greater than 80 years old,or greater than 90 years old. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is asubject with atypical hyperplasia of the breast, ductal carcinoma insitu (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS),lobular neoplasia, or a stage 0 growth or lesion of the breast (e.g.,stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1 (mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder.” A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of the presentinvention that has been or will be tested in one or more in vitro or invivo biological assays, in order to determine if that compound is likelyto elicit a desired biological or medical response in a cell, tissue,system, animal or human that is being sought by a researcher orclinician. A candidate compound is a pharmaceutical compositioncomprising (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than containing less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients. The biologicalor medical response can be the treatment of cancer. The biological ormedical response can be treatment or prevention of a cell proliferativedisorder. In vitro or in vivo biological assays can include, but are notlimited to, enzymatic activity assays, electrophoretic mobility shiftassays, reporter gene assays, in vitro cell viability assays, and theassays described herein.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an active compound. For example, cancer monotherapy with oneof the compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to asubject in need of treatment of cancer. Monotherapy may be contrastedwith combination therapy, in which a combination of multiple activecompounds is administered, preferably with each component of thecombination present in a therapeutically effective amount. In oneaspect, monotherapy with a compound of the present invention is moreeffective than combination therapy in inducing a desired biologicaleffect.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention to alleviate the symptoms or complications of a disease,condition or disorder, or to eliminate the disease, condition ordisorder.

The compounds of the present invention can also be used to prevent adisease, condition or disorder. As used herein, “preventing” or“prevent” describes reducing or eliminating the onset of the symptoms orcomplications of the disease, condition or disorder.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression.” Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively on a canceror precancerous cell but not on a normal cell. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively to modulateone molecular target (e.g., c-met) but does not significantly modulateanother molecular target (e.g., Protein Kinase C). The invention alsoprovides a method for selectively inhibiting the activity of an enzyme,such as a kinase. Preferably, an event occurs selectively in populationA relative to population B if it occurs greater than two times morefrequently in population A as compared to population B. An event occursselectively if it occurs greater than five times more frequently inpopulation A. An event occurs selectively if it occurs greater than tentimes more frequently in population A; more preferably, greater thanfifty times; even more preferably, greater than 100 times; and mostpreferably, greater than 1000 times more frequently in population A ascompared to population B. For example, cell death would be said to occurselectively in cancer cells if it occurred greater than twice asfrequently in cancer cells as compared to normal cells.

A compound of the present invention or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can modulatethe activity of a molecular target (e.g., c-met). Modulating refers tostimulating or inhibiting an activity of a molecular target. Preferably,a compound of the present invention modulates the activity of amolecular target if it stimulates or inhibits the activity of themolecular target by at least 2-fold relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound. More preferably, a compound of the present inventionmodulates the activity of a molecular target if it stimulates orinhibits the activity of the molecular target by at least 5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, at least 100-foldrelative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, does notsignificantly modulate the activity of a molecular target if theaddition of the compound does not stimulate or inhibit the activity ofthe molecular target by greater than 10% relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a kinase isozyme alpha in comparison to a kinase isozymebeta). Preferably, a compound of the present invention demonstrates aminimum of a four fold differential, preferably a ten fold differential,more preferably a fifty fold differential, in the dosage required toachieve a biological effect. Preferably, a compound of the presentinvention demonstrates this differential across the range of inhibition,and the differential is exemplified at the IC₅₀, i.e., a 50% inhibition,for a molecular target of interest.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof can result in modulation (i.e.,stimulation or inhibition) of an activity of a kinase of interest.

The present invention provides methods to assess biological activity ofa composition comprising (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than containing less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;(b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation; or (c) aform 2 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation.

In one method, an assay based on enzymatic activity can be utilized. Inone specific enzymatic activity assay, the enzymatic activity is from akinase. As used herein, “kinase” refers to a large class of enzymeswhich catalyze the transfer of the γ-phosphate from ATP to the hydroxylgroup on the side chain of Ser/Thr or Tyr in proteins and peptides andare intimately involved in the control of various important cellfunctions, perhaps most notably: signal transduction, differentiation,and proliferation. There are estimated to be about 2,000 distinctprotein kinases in the human body, and although each of thesephosphorylate particular protein/peptide substrates, they all bind thesame second substrate ATP in a highly conserved pocket. About 50% of theknown oncogene products are protein tyrosine kinases (PTKs), and theirkinase activity has been shown to lead to cell formation. Preferably,the kinase assayed is a tyrosine kinase.

A change in enzymatic activity caused by compounds of the presentinvention can be measured in the disclosed assays. The change inenzymatic activity can be characterized by the change in the extent ofphosphorylation of certain substrates. As used herein, “phosphorylation”refers to the addition of phosphate groups to a substrate, includingproteins and organic molecules; and, plays an important role inregulating the biological activities of proteins. Preferably, thephosphorylation assayed and measured involves the addition of phosphategroups to tyrosine residues. The substrate can be a peptide or protein.

In some assays, immunological reagents, e.g., antibodies and antigens,are employed. Fluorescence can be utilized in the measurement ofenzymatic activity in some assays. As used herein, “fluorescence” refersto a process through which a molecule emits a photon as a result ofabsorbing an incoming photon of higher energy by the same molecule.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof can result in modulation (i.e.,stimulation or inhibition) of an activity of c-Met. As used herein, anactivity of c-Met refers to any biological function or activity that iscarried out by c-Met. For example, a function of c-Met includesphosphorylation of downstream target proteins. Other functions of c-Metinclude autophosphorylation, binding of adaptor proteins such as Gab-1,Grb-2, Shc, SHP2 and c-Cbl, and activation of signal transducers such asRas, Src, PI3K, PLC-γ, STATs, ERK1 and 2 and FAK.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof can result in modulation (i.e.,stimulation or inhibition) of an activity of ERK 1 or ERK 2, or both. Asused herein, an activity of ERK 1 or ERK 2 refers to any biologicalfunction or activity that is carried out by ERK 1 or ERK 2. For example,a function of ERK 1 or ERK 2 includes phosphorylation of downstreamtarget proteins.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

As used herein, “a cell cycle checkpoint pathway” refers to abiochemical pathway that is involved in modulation of a cell cyclecheckpoint. A cell cycle checkpoint pathway may have stimulatory orinhibitory effects, or both, on one or more functions comprising a cellcycle checkpoint. A cell cycle checkpoint pathway is comprised of atleast two compositions of matter, preferably proteins, both of whichcontribute to modulation of a cell cycle checkpoint. A cell cyclecheckpoint pathway may be activated through an activation of one or moremembers of the cell cycle checkpoint pathway. Preferably, a cell cyclecheckpoint pathway is a biochemical signaling pathway.

As used herein, “cell cycle checkpoint regulator” refers to acomposition of matter that can function, at least in part, in modulationof a cell cycle checkpoint. A cell cycle checkpoint regulator may havestimulatory or inhibitory effects, or both, on one or more functionscomprising a cell cycle checkpoint. A cell cycle checkpoint regulatorcan be a protein or not a protein.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,(2003) Proc Natl Acad Sci USA. 100(5): 2674-8. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof is not significantly cytotoxic to normal cells. Atherapeutically effective amount of a compound is not significantlycytotoxic to normal cells if administration of the compound in atherapeutically effective amount does not induce cell death in greaterthan 10% of normal cells. A therapeutically effective amount of acompound does not significantly affect the viability of normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. In anaspect, cell death occurs by apoptosis.

Contacting a cell with a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, can induce or activate cell death selectively in cancercells. Administering to a subject in need thereof a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce or activate celldeath selectively in cancer cells. Contacting a cell with a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce cell deathselectively in one or more cells affected by a cell proliferativedisorder. Preferably, administering to a subject in need thereof acompound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, induces celldeath selectively in one or more cells affected by a cell proliferativedisorder.

The present invention relates to a method of treating or preventingcancer by administering a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof to a subject in need thereof, where administration ofthe compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof results in oneor more of the following: accumulation of cells in G1 and/or S phase ofthe cell cycle, cytotoxicity via cell death in cancer cells without asignificant amount of cell death in normal cells, antitumor activity inanimals with a therapeutic index of at least 2, and activation of a cellcycle checkpoint. As used herein, “therapeutic index” is the maximumtolerated dose divided by the efficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3d ed.), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention

As used herein, “combination therapy” or “co-therapy” includes theadministration of a compound of the present invention and at least asecond agent as part of a specific treatment regimen intended to providethe beneficial effect from the co-action of these therapeutic agents.The beneficial effect of the combination includes, but is not limitedto, pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected). “Combination therapy” may be, but generally isnot, intended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentinvention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, wherein each therapeuticagent is administered at a different time, as well as administration ofthese therapeutic agents, or at least two of the therapeutic agents, ina substantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, may beadministered in combination with a second chemotherapeutic agent. Thesecond chemotherapeutic agent can be a taxane, an aromatase inhibitor,an anthracycline, a microtubule targeting drug, a topoisomerase poisondrug, a targeted monoclonal or polyconal antibody, an inhibitor of amolecular target or enzyme (e.g., a kinase inhibitor), or a cytidineanalogue drug. Preferably, the chemotherapeutic agent can be, but notrestricted to, tamoxifen, raloxifene, anastrozole, exemestane,letrozole, HERCEPTIN® (trastuzumab), GLEEVEC® (imatanib), TAXOL®(paclitaxel), cyclophosphamide, lovastatin, minosine, araC,5-fluorouracil (5-FU), methotrexate (MTX), TAXOTERE® (docetaxel),ZOLADEX® (goserelin), vincristin, vinblastin, nocodazole, teniposide,etoposide, GEMZAR® (gemcitabine), epothilone, navelbine, camptothecin,daunonibicin, dactinomycin, mitoxantrone, amsacrine, doxorubicin(adriamycin), epirubicin or idarubicin. The second chemotherapeuticagent can be a cytokine such as G-CSF (granulocyte colony stimulatingfactor). A compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, maybe administered in combination with radiation therapy. A compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, may be administered incombination with standard chemotherapy combinations such as, but notrestricted to, CMF (cyclophosphamide, methotrexate and 5-fluorouracil),CAF (cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycinand cyclophosphamide), FEC (5-fluorouracil, epirubicin, andcyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, andpaclitaxel), or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil andprednisone)

A compound of the invention, or a pharmaceutically acceptable salt,prodrug, metabolite, polymorph or solvate thereof, may be administeredwith an inhibitor of an enzyme, such as a receptor or non-receptorkinase. Receptor and non-receptor kinases of the invention are, forexample, tyrosine kinases or serine/threonine kinases. Kinase inhibitorsof the invention are small molecules, polynucleic acids, polypeptides,or antibodies.

Exemplary tyrosine kinases include, but are not limited to, Bevacizumab(targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux(targets Erb1), Imatinib/Gleevec (targets Bcr-Abl), Trastuzumab (targetsErb2), Gefitinib/Iressa (targets EGFR), Ranibizumab (targets VEGF),Pegaptanib (targets VEGF), Erlotinib/Tarceva (targets Erb1), Nilotinib(targets Bcr-Abl), Lapatinib (targets Erb1 and Erb2/Her2),GW-572016/lapatinib ditosylate (targets HER2/Erb2), Panitumumab/Vectibix(targets EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targetsincluding RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166(targets EGFR), Canertinib/CI-1033 (targets EGFR),Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200(targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR),PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targetsVEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targetsFLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targetsSRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targetsJAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targetsJAK), Sorafinib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-β, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src),AC-220 (targets Flt3), AC-480 (targets all HER proteins, “panHER”),Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), Denosumab(targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534(multiple targets including Flt3).

Exemplary serine/threonine kinases include, but are not limited to,Rapamune (targets mTOR/FRAP1), Deforolimus (targets mTOR),Certican/Everolimus (targets mTOR/FRAP1), AP23573 (targets mTOR/FRAP1),Eril/Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK),Seliciclib/CYC202/Roscovitrine (targets CDK), SNS-032/BMS-387032(targets CDK), Ruboxistaurin (targets PKC), Pkc412 (targets PKC),Bryostatin (targets PKC), KAI-9803 (targets PKC), SF1126 (targets PI3K),VX-680 (targets Aurora kinase), Azd1152 (targets Aurora kinase),Arry-142886/AZD-6244 (targets MAP/MEK), SCID-469 (targets MAP/MEK),GW681323 (targets MAP/MEK), CC-401 (targets JNK), CEP-1347 (targetsJNK), and PD 332991 (targets CDK).

Preferred combinatorial therapies include, but are not limited to, (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients, administered incombination with Erlotinib, (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients administered incombination with gemcitabine, and (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients administered incombination with sorafinib. In certain embodiments, a subject or patientreceives a combination of Erlotinib, administered as 150 mg once daily,in combination with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,administered as 360 mg twice daily. In other embodiments, a subject orpatient receives a combination of gemicitabine, administered by 1000mg/m² intravenous infusion over 30 minutes once weekly, in combinationwith(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,administered as 360 mg by mouth twice a day or 120 mg by mouth twice aday continuously. In another embodiment, a subject or patient receives acombination of sorafinib, administered as 200 mg twice daily, incombination with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,administered as 360 mg twice daily. Preferred dosage forms for(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinclude, but are not limited to, a capsule and a tablet.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising (a)(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehaving a chiral purity greater than 99% as determined by HPLC andcontaining less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a pharmaceutically acceptable carrier; (b) a form 1 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients; or (c) a form 2polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation, and one ormore pharmaceutically acceptable carriers or excipients.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, (e.g., intravenous, intradermal,subcutaneous), oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not so high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount,” as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is cancer. In another aspect, thedisease or condition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered daily, every 3 to 4days, every week, or once every two weeks depending on half-life andclearance rate of the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringeability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and/or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch, pregelatinized starch, or lactose,a disintegrating agent such as croscarmellose sodium, sodium starchglycolate, sodium carboxymethyl starch, alginic acid, Primogel,crospovidone, or corn starch; a lubricant such as magnesium stearate,stearic acid, sodium stearyl fumarate, or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser,which contains a suitable propellant, e.g., hydrofluoroalkanes,chlorofluorocarbons, carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid ormixtures or copolymers of polyesters. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). In preferred applications, the dosage can beapproximately 400 milligrams twice daily (b.i.d.). In specificapplications, the dosage is 360 milligrams (mg) twice daily (b.i.d.).More preferably, dosage form is a capsule or tablet and is administeredas two or three capsules or tablets with a combined dosage of 360milligrams (mg). This dosage form is administered twice daily for atotal dose of 720 milligrams (mg). An effective amount of apharmaceutical agent is that which provides an objectively identifiableimprovement as noted by the clinician or other qualified observer. Forexample, regression of a tumor in a patient may be measured withreference to the diameter of a tumor. Decrease in the diameter of atumor indicates regression. Regression is also indicated by failure oftumors to recur after treatment has stopped. As used herein, the term“dosage effective manner” refers to amount of an active compound toproduce the desired biological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present invention can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., an acetate, propionate or other ester.

The compounds of the present invention can also be prepared as prodrugs,for example, pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs in thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxyl, amino,sulfhydryl, carboxy or carbonyl group is bonded to any group that, maybe cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, hydroxy, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxyl functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, New York-Oxford (1985).

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

Other features and advantages of the present invention are apparent fromthe different examples. The provided examples illustrate differentcomponents and methodology useful in practicing the present invention.The examples do not limit the claimed invention. Based on the presentdisclosure the skilled artisan can identify and employ other componentsand methodology useful for practicing the present invention.

EXAMPLES Example 1

The present example describes the preparation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione.

Lilolidine [CAS 102280-97-7] (70 kg) (Compound 4 in Scheme I) wascharged to an appropriately cleaned and dry reactor vessel followed bymethyl tert-butyl ether (MTBE) (375 kg). Lilolidine may be purchasedcommercially or prepared as described in U.S. Patent ApplicationPublication No. 2006/0223760. The resulting batch was agitated for aminimum of 10 minutes at 15-25° C. A solution of oxalyl chloride (56.6kg) in MTBE (370 kg) was prepared in a separate vessel. The lilolidinesolution was then added to the oxalyl chloride solution at such a rateto maintain the temperature below 32° C. The vessel was rinsed withadditional MTBE (162 kg) and added to the reaction mixture. The batchwas stirred at 15-32° C. for a minimum of two hours prior to analysis byHPLC. When the reaction was determined to be complete, methanol (90 kg)was added, and the batch was stirred for a minimum of two hours. Whenthe reaction was determined to be complete by HPLC analysis, the batchwas distilled to approximately 80 gallons. It is not necessary toisolate the intermediate 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)oxoacetic acid methyl ester (Compound 5 in Scheme I). Tetrahydrofuran(THF) (840 kg) was added to the batch and the volume was again reducedto approximately 80 gallons by distillation. This solvent swap processwas continued until the amount of MTBE present in the batch was <1% byweight. A new vessel was charged with indole-3-acetamide [CAS 879-37-8](61.3 kg) (Compound 5a in Scheme I) followed by THF (840 kg). Theresulting solution was then charged to the batch at a rate such that thetemperature was maintained at 15-25° C. The vessel containing thesolution of indole-3-acetamide was rinsed with THF (140 kg), and therinse was added to the batch. The empty vessel was then charged withpotassium tert-butoxide solution (1.6 M in THF, 581 kg) and THF (350kg). The solution was also added to the batch, and the resultingsolution was stirred at 20-32° C. for a minimum of three hours. When thestarting material had been consumed as confirmed by HPLC analysis,aqueous HCl (conc., 273 kg) was added at such a rate that thetemperature was maintained below 50° C. The batch was stirred at 40-50°C. for a minimum of 30 minutes.

When the reaction had been determined to be complete by HPLC analysis,aqueous ammonium hydroxide solution (conc.) was added, while maintainingthe reaction temperature below 40° C., until the pH of the mixture was9-10. Following the addition of ethyl acetate (EtOAc) (462 kg) andagitation of the batch, the layers were separated. The organic layer waswashed with brine (182 kg NaCl and 1022 kg water). The resulting organicsolution was distilled to approximately one third of the startingvolume. Ethanol (2B, 1120 kg) was added, and the distillation wascontinued to reduce the batch volume to approximately 240 gallons.Ethanol (2B, 1120 kg) was again added, and the volume reduced to 240gallons. Water (1400 kg) was then added to the batch to induceprecipitation of the product. The batch was agitated for a minimum oftwo hours, and the solids were isolated by filtration. The solids werethen taken up in dichloromethane (DCM) (840 kg), and heptanes (442 kg)were added to purify the product. Following agitation of the batch forat least two hours, the product was isolated by filtration. Followingconditioning on the filter, approximately 115 kg (88%) of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione(Compound 6 in Scheme I) was isolated as a red powder. This conversionof Compound 4 to Compound 6 is shown in Scheme II.

Example 2

The present example describes the preparation of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Palladium hydroxide (20 wt % Pd on carbon, 11.5 kg) was charged to aproperly prepared reaction vessel. THF (340 kg) was added to produce aslurry, and the catalyst was prereduced with hydrogen (50-75 psi).Compound 6 (115 kg) was charged to an empty vessel followed by THF (353kg). The resulting mixture was stirred until dissolution was complete.The solution of Compound 6 was then transferred to the slurry of thecatalyst. A solution of potassium tert-butoxide (1.6 M in THF, 36 kg)was charged to an empty reactor followed by THF (21 kg). This resultingsolution was also transferred to the reaction mixture followed by anadditional rinse with THF (340 kg). The batch was then heated to 45-55°C. under 65-80 psi of hydrogen. It is not necessary to isolate theintermediate cis racemate(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 7 in Scheme I).

When the reaction was determined to be complete by HPLC analysis, thebatch was filtered through Celite to remove the catalyst and dilutedwith isopropylacetate (iPrOAc) (810 kg). The organic solution was washedwith aqueous HCl (28 kg conc. HCl, 290 kg water). This process wasrepeated a second time. The organic solution was then washed with brine(580 kg) prior to being concentrated to approximately 300 gallons bydistillation. iPrOAc (1690 kg) was added, and the batch was distilled toapproximately 400 gallons. The distillation process was repeated byadding iPrOAc (1000 kg) until the THF content was <2% by weight in thesolution. Heptanes (2000 kg) were then added to induce precipitation ofthe product. The crude(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 8 in Scheme I) was isolated by filtration and conditioned toafford 111 kg (95%) having an HPLC purity of ˜96%. This material alsocontained 1.7% iPrOAc and 6.3% heptane. This material was confirmedthrough laboratory scale “use” testing to be of sufficient purity toproceed with preparation of the(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane (DCM) and resolution by multicolumn chromatography (MCC)or for direct use in crystallization of the diastereomeric salt, asdescribed in further detail in the following examples.

Example 3

The present example describes preparation of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneDCM.

It is preferred to chemically purify Compound 8 by production ofcrystalline(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneDCM (Compound 8 DCM in Scheme I) prior to the process of enantiomericresolution by MCC.

In one example, the reaction vessel was charged with methanol (125 kg)and Compound 8 (52.5 kg) and the resulting mixture was heated to 55-65°C. DCM (557 kg) was charged to a clean reactor. The solution of Compound8 was then transferred to the reactor containing DCM through an inlinefilter. The reactor was rinsed with DCM (134 kg) which was alsotransferred through the filter to the reactor containing the reactionmixture. The batch was agitated for a minimum of 30 minutes prior to theintroduction of Compound 8 DCM seeds (0.1 kg). The batch was stirred forat least four hours and then sampled to estimate the extent ofcrystallization. When the filtrate concentration of Compound 8 was below65 mg/mL, heptanes (718 kg) were added and the batch was stirred for atleast one hour. The batch was then cooled to 0-5° C., and the productwas isolated by filtration. The solids were conditioned on the filterand dried in a vacuum tray drier at 45-55° C. for at least four hours.Compound 8 DCM (48.3 kg, 92%, HPLC 99.0%) was isolated as a tan solid.The conversion of Compound 6 to Compound 8 DCM described in examples 2and 3 are shown in Scheme III.

Example 4

The present example describes the chiral resolution of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneDCM by MCC and isolation of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

A feed solution for multicolumn chromatography (MCC) is prepared bydissolving Compound 8 DCM in methanol. The resulting DCM/methanolco-solvent is distilled until the residual level of DCM in the feedsolution reaches a level acceptable for contact with the ChiralStationary Phase (CSP), i.e., <0.1% by weight. The batch is diluted in amixture of methanol/Acetonitrile (9:1) to a concentration of 50 mg/mLand introduced to the chromatographic system. Chiralpak AZ (CSP) can beutilized. The raffinate is monitored by chiral HPLC analysis andchromatographic parameters are tuned to obtain >99% chiral purity of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 10 in Scheme I). Raffinate is pooled and concentratedbatchwise as the separation proceeds. The collected raffinate is takento a larger reactor and the volume is reduced. Compound 10 is thenisolated by crystallization.

Example 5

The present example describes batchwise generation of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom the undesired enantiomer,(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

In some instances, for instance when resolution is achieved using MCC,it is desirable to isolate and racemize the undesired enantiomer,(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Compound 9 in Scheme I), in a batchwise manner

In one example of the process to generate crude Compound 8 from isolatedCompound 9, the undesired enantiomer (6.46 kg) was charged to a 100-Lreactor. Ethanol (60 L) was charged followed by the addition of solidNaOH (1.05 kg, 1.5 equiv). The resulting slurry was heated to 65° C. for13 hours and then allowed to cool to ambient temperature over 4 hours.The mixture was sampled and analyzed by chiral HPLC showing a 54/46ratio of enantiomers. The chemical purity was determined to be 94.6%(AUC) by HPLC. The slurry was then polish filtered through Celite, andthe pad was rinsed with ethanol (13 L). The material that was removedwas a brown film of solids with no large chunks. The solution was thenrecharged to the 100-L reactor and 2 M HCl (13.1 L, 1.5 equiv) was addedover 35 minutes. During the addition, a thin slurry formed. The mixturewas then stirred at ambient temperature (after 5 hours a thick lightorange slurry was obtained). Water (25 L) was then added over 45minutes, and the mixture was stirred for 2 hours. An aliquot was removedand filtered. The batch was then heated to 60° C. and stirred for 8hours, and then allowed to slowly cool to improve the filtrationproperties of the solids. Crude Compound 8 [5.12 kg, 75.7% (accounts for4.5 wt % ethanol)] was isolated with chemical purity of 99.39% (AUC) byHPLC. In this case the purity was sufficient to further process thismaterial by MCC. The purity can be enhanced, if necessary, throughformation of the DCM.

The chiral resolution of Compound 8 DCM to Compound 10 described inExample 4 and the recycling of Compound 9 to Compound 8 described inExample 5 are shown in Scheme IV.

Example 6

The present example describes the chiral resolution by diastereomericsalt formation and isolation of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

As an alternative to chiral resolution by MCC, a resolution of theenantiomers, Compound 10 and Compound 9, can also be achieved by thepreferential formation of a diastereomeric salt of Compound 10.

In one example, crude Compound 8 can be converted to(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine(Compound 10.(1S,2S)-(+)-pseudoephedrine in Scheme I) using thefollowing representative procedure. (1S,2S)-(+)-pseudoephedrine (601 g,0.6 equiv) is dissolved in acetonitrile (CH₃CN) (11.2 L, 5 vol) at about50° C. and stirred for approximately 30 minutes past dissolution. CrudeCompound 8 [2.24 kg, 96.4% (AUC) by HPLC] is dissolved in CH₃CN (11.2 L,5 vol) at about 50° C. and then polish filtered through a pad of Celite.The Compound 8 solution is added via a drop tank to the(1S,2S)-(+)-pseudoephedrine solution over 20-30 minutes at about 50° C.Once the resulting solution begins to crystallize, shortly aftercompletion of the Compound 8 addition, the mixture is stirred until itslowly cools to ambient temperature, then stirring is continuedovernight (about 11 h total of cooling and stirring). The beige granularsolids are filtered through an 18″ stainless steel nutche filter andrinsed/re-slurried using CH₃CN (7 L, 3.2 vol). The slurry is filtered,and the solids are slurried again with CH₃CN (5 L, 2.3 vol). Afterdrying on the nutche filter for 1 hour a sample is analyzed by HPLC. Ifthe solids contain a higher than desired amount of the undesireddiastereomeric salt, the solids can be re-slurried using CH₃CN and driedbefore repeating the analysis. The solids are then dried under hot N₂(51° C.) overnight. Typically, analysis of the Compound10.(1S,2S)-(+)-pseudoephedrine shows an overall chemical purity of >99%(AUC) by HPLC and a chiral purity of >99% Compound 10. Using ¹H NMRanalysis, a typical sample is estimated to contain <0.5 wt % CH₃CN.

The Compound 10.(1S,2S)-(+)-pseudoephedrine can then be converted toCompound 10 by treatment with acid and crystallization from methanol orethanol. A representative procedure is as follows: Compound10.(1S,2S)-(+)-pseudoephedrine (50 g) is slurried inMethyltetrahydrofuran (MeTHF) (500 mL) and water (250 mL). 1 M HCl (110mL) is added to the mixture to reach a final pH of 1.6. The resultingsolution with a slight amount of solids is stirred for about one hour todissolve the solids. The organic and aqueous layers are then separated.The organic layer is washed with a water/brine solution (1:1, 250 mL),separated, and 2-B ethanol (1000 mL) is added. The solution is thenconcentrated down to 200 mL and analyzed for MeTHF content (4.8 wt %).The solution is polish filtered, and Compound 10 seeds (0.2 g) are added(T=25° C.). The mixture is stirred for about three days with samplesbeing taken periodically and analyzed for Compound 10 remaining in themother liquor. The resulting slurry is filtered, and the solids arewashed with 2-B ethanol (70 mL). The solids are dried for about threehours in a vacuum oven at about 60° C. to give Compound 10 [28.84 g,82.7%, 99.60% (AUC) by HPLC, 0.54 wt % ethanol, 0.08 wt % MeTHF] as abeige solid.

The diasteromeric salt resolution of Compound 8 to Compound 10 describedin Example 6 and the recycling of Compound 9 to Compound 8 described inExample 5 are shown in Scheme V.

Example 7

The present example describes chiral separation by dynamic kineticresolution (DKR).

As an alternative to chiral resolution by MCC or a traditional kineticresolution by diastereomeric salt formation, a dynamic kineticresolution (DKR) of the enantiomers Compound 10 and Compound 9 can alsobe achieved by the preferential formation of a diastereomeric salt ofCompound 10 with simultaneous in situ racemization of Compound 9.

In this process, crude Compound 8 can be converted to Compound10.(1S,2S)-(+)-pseudoephedrine using the following representativeprocedure. Crude Compound 8 (1.25 kg, 96% AUC, 9.1 wt % solvent content)and (1S,2S)-(+)-pseudoephedrine (559 g, 1.0 equiv) were slurried in2B-ethanol (11.25 L, 9 vol) and heated to 50° C. for 3 hours. The slurrywas treated with 21 wt % NaOEt in methanol or ethanol (110 g, 0.1 equiv)and heated to 50° C. After 40 hours, the mixture was quenched by adding1 M HCl (338 mL, 0.1 equiv) in water (786 mL, ˜10% water relative toethanol) over 5 minutes. The mixture was stirred at 50° C. for 1 hour,and then allowed to cool to ambient temperature over 0.5 hours. Theslurry was stirred at room temperature for another 3 hours, and thenfiltered. The solids were washed with 10% water/2B-ethanol (3.75 L, 3vol) and dried in a vacuum oven (50° C., 2 trays) for 18 hours. Thesolids were analyzed and showed Compound 10.(1S,2S)-(+)-pseudoephedrine[1.22 Kg, 74%, 99.3% (AUC) by HPLC, 99.2% (AUC) by chiral HPLC]. Thisprocess was carried out on 20 kg of Compound 8 to afford Compound10.(1S,2S)-(+)-pseudoephedrine [18.9 kg, 70%, %, 98.8% (AUC) by HPLC,99.1% (AUC) by chiral HPLC].

The dynamic kinetic resolution of Compound 8 to Compound 10 via Compound10.(1S,2S)-(+)-pseudoephedrine is shown in Scheme VI.

Example 8

The present example describes chiral separation by dynamic kineticresolution (DKR).

As an alternative to chiral resolution by MCC or a traditional kineticresolution by diastereomeric salt formation, a dynamic kineticresolution (DKR) of the enantiomers Compound 10 and Compound 9 can alsobe achieved by the preferential formation of a diastereomeric salt ofCompound 10 with simultaneous in situ racemization of Compound 9.

In this process, crude Compound 8 (111.4 kg), Pd scavenger resin(PL-TMT-MP, 3.5 kg), and methanol (1507 L) were added to a container andheated to 45° C. for at least 16 hours. Upon completion of the reaction,the mixture was filtered to remove the Pd scavenger resin. The containerwas rinsed with methanol (104 L). The combined filtrate was distilled to˜600 L. (1S,2S)-(+)-pseudoephedrine (51.4 kg) and methanol (416 L) wereadded to the filtrate. The resulting solution was heated at 45° C. for3-4 hours. Solids were precipitated and analyzed by HPLC to ensure thereaction was proceedingly selective. A solution of sodium methoxide inmethanol (21% wt, 6.1 kg) was then added followed by methanol (11 L),and the resulting mixture was heated at 45° C. for an additional 18hours. The reaction mixture was analyzed by HPLC for completion of thecrystallization. Upon completion of the crystallization, the reactionmixture was treated with HCl solution (3.5 kg) to neutralize the base.The resulting mixture was cooled to ambient temperature and agitated fora minimum of 3 hours. The solids were then isolated by filtration, andthe filter cake was washed with a solution of methanol and water (411 Lto 46 L, respectively). The filter cake was colorless (if significantcolor persisted in the solids, the washing was repeated to remove thecolor). The solids were dried in a filter dryer at 55° C. under vacuumfor a minimum of 8 hours and released for use in the next step asCompound 10.(1S,2S)-(+)-pseudoephedrine (101 kg).

Example 9

The present example describes a slurry method to prepare(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Compound 10.(1S,2S)-(+)-pseudoephedrine (100.7 kg) and a solution ofaqueous HCl (1M, 250 kg) and methanol (240 kg) were mixed, and theresulting slurry was heated to 50° C. and stirred for at least 2 hours.Upon completion of the reaction, the slurry was cooled to ambienttemperature and stirred for a minimum of 1 hour. The solids wereisolated by filtration, washed with a 50% solution of aqueous methanol(200 L), and dried under vacuum at 65° C. for at least 6 hours. Thecrude Compound 10 was dissolved in THF (96 kg) by heating the mixture to50° C. The resulting solution was polish filtered, followed by theaddition of methanol (200 kg). The solution was then concentratedazeotropically and atmospherically by distillation to reduce the THFcontent. Once the volume of the solution was reduced to 250 L,additional methanol (200 L) was added, and the concentration process wasrepeated. This process was repeated until the THF content was reduced toless than 5% (vol./vol.). During the addition of methanol, one or morecrystals of Compound 10 (300 g) were also introduced in order tofacilitate crystallization. The crystals were isolated by filtration,washed with additional 50% aqueous methanol, and dried under vacuum at65° C. for at least 12 hours. Crude Compound 10 (60.6 kg) was isolatedas a red-brown solid.

Example 10

The present example describes a slurry method to prepare Form 1polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Crude Compound 10 was prepared as described in Example 9. The crudeCompound 10 was dissolved in THF (96 kg) by heating the mixture to 50°C. The resulting solution was polish filtered, followed by the additionof methanol (200 kg) and Form 1 of Compound 10 as seeds to impartpolymorphic control of the crystallization process. Seeds to impartpolymorphic control were taken from representative batches in whichcharacterization data had confirmed that the crystalline material was ofthe desired polymorphic form. The solution was then concentratedazeotropically and atmospherically by distillation to reduce the THFcontent. Once the volume of the solution was reduced to 250 L,additional methanol (200 L) was added, and the concentration process wasrepeated. This process was repeated until the THF content was reduced toless than 5% (vol./vol.). The temperature of the solution was thenreduced to 50° C. and stirred for at least 4 hours. Aliquots wereremoved to confirm formation of the desired polymorph. If required, thepolymorph can be redissolved in THF (30% of batch volume), polishfiltered, concentrated and seeded to obtain the desired polymorph. Whenthe desired polymorph was obtained, a solution of 50% aqueous methanolwas added at 50° C., and the solution was agitated for an additional 2-3hours. The solution was then cooled to ambient temperature and held forat least 2 hours to allow crystallization. Upon completion, the crystalswere isolated by filtration, washed with additional 50% aqueousmethanol, and dried under vacuum at 65° C. for at least 12 hours.Polymorphic Form 1 of Compound 10 (60.6 kg) was isolated as a red-brownsolid.

Form 2 polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecan also be prepared with the above method if the Form 1 seed crystal isreplaced by the Form 2 seed crystal.

Example 11

Example 11 describes a method to generate XRPD diffractograms ofpolymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

X-Ray Powder Diffraction patterns were collected on a Siemens D5000diffractometer using Cu Kα radiation (40 kV, 40 mA), 0-0 goniometer,divergence of V20 and receiving slits, a graphite secondarymonochromator and a scintillation counter. The instrument performance ischecked using a certified Corundum standard (NIST 1976). The softwareused for data collection was Diffrac Plus XRD Commander v2.3.1 and thedata were analyzed and presented using Diffrac Plus EVA v 11.0.0.2 or v13.0.0.2.

Powder samples were prepared as flat plate specimens. Approximately 35mg of the sample was gently packed into a cavity cut into polished,zero-background (510) silicon wafer. The sample was rotated in its ownplane during analysis. The details of the data collection are:

-   -   Angular range: 2 to 42° 2θ    -   Step size: 0.05° 2θ    -   Collection time: 4 s/step.

Example 12

Example 12 describes a method to generate XRPD diffractograms ofpolymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

High resolution X-Ray Powder Diffraction patterns were collected on aBruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), 0-20goniometer, and a divergence of V4 and receiving slits, aGe-monochromator and a Lynxeye detector. The instrument performance ischecked using a certified Corundum standard (NIST 1976). The softwareused for data collection was Diffrac Plus XRD Commander v2.5.0 and thedata were analyzed and presented using Diffrac Plus EVA v 11.0.0.2 or v13.0.0.2.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. Approximately 100 mg of the sample was gently packedinto a circular cavity cut into polished, zero-background (510) siliconwafer. The sample was rotated in its own plane during analysis. Thedetails of the data collection for the generic procedure are:

-   -   Angular range: 2 to 42° 2θ    -   Step size: 0.05° 2θ    -   Collection time: 5 s.step⁻¹

Example 13

Example 13 describes XRPD patterns from polymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

Two non-solvated polymorphs of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione:Form 1 and Form 2 (See, FIGS. 12 and 13).

Form 1 shows a XRPD pattern comprising 2θ values in degrees of 8.2, 10.8and 14.1. (See, FIG. 12 and Table 1)

Form 2 shows a XRPD patter comprising 2θ values in degrees of 6.5, 9.9and 12.0. (See, FIG. 13 and Table 1)

TABLE 1 8.2 10.8 14.1 Total 6.5 9.9 12.0 Total Form 1 area area areaarea Form 2 area area area area 1 3.925 14.08 6.646 24.651 1 6.433 5.28410.40 22.117 2 3.866 13.98 6.608 24.454 2 6.437 5.365 10.51 22.312 33.847 14.03 6.625 24.502 3 6.504 5.341 10.49 22.335 4 3.880 13.99 6.55624.426 4 6.467 5.321 10.44 22.228 5 3.889 13.88 6.619 24.388 5 6.4845.331 10.50 22.315 6 3.854 13.92 6.586 24.360 6 6.502 5.281 10.38 22.163mean 3.88 14.0 6.61 24.464 mean 6.47 5.32 10.45 22.245 RSD (%) 0.73 0.520.48 0.43 RSD (%) 0.00 0.00 0.01 0.02

Example 14

Form 1 is more thermodynamically stable than Form 2 as determined ininterconversion experiments. Form 1 is orthorhombic and contains fourmolecules of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneper cell. Form 1 has a melting point of ˜218° C. recorded in DSC (See,FIG. 16).

Form 2 is comprised of birefringent rod-like crystals. No discernablechange in crystalline form or purity was observed upon storage ofmultiple batches of Form 2 at 40° C./75% RH for up to six months and 25°C./60% RH for up to 12 months. The space group of Form 2 was either P2₁or P2: there are two molecules of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein the unit cell (one molecule in the asymmetric unit), and there is noexcess volume in the unit cell that is occupied by a solvent molecule.Form 2 has a melting point of ˜216° C. recorded in DSC (See, FIG. 16).

Upon scale-up of the Compound 10 process to >5 kg production as a singlebatch, Form 1 was spontaneously produced. Infra-red (IR) and X-raypowder diffraction (XRPD) were determined to be effective analyticaltools for the identification and differentiation of the two forms (See,FIGS. 14 and 15). The process was readily controlled via seedingdemonstrated at a 7-kg scale.

The two polymorphs can be interconverted when proper conditions are usedto control the crystallization process. Form 2 can be obtained bydissolving Form 1 in methanol, seeding with Form 2 and evaporating themixture to dryness by rotary evaporation. The successful conversion wasdetermined to be Form 2 by IR analysis and corroborated by XRPDanalysis.

Example 15

The IR spectrum of Form 1 displayed an intense vibration centered at˜3300 cm⁻¹ that was not observed in Form 2 (FIG. 15, Panel A), whereasthe IR spectrum of Form 2 showed a relatively intense peak centered at˜800 cm⁻¹ which was not observed in Form 1 (FIG. 15, Panel B).

Form 1 and Form 2 have different equilibrium solubilities: Form 2=0.61mg/mL and Form 1=0.51 mg/mL. FIG. 17, Panel A shows the solubility ofForms 1 and 2 in methanol from 0-70° C. FIG. 17, Panel B shows theintrinsic solubility of Forms 1 and 2 in 50 mM pH 6.8 phosphatebuffer/1% SLS.

Example 16

Exponentially growing MDA-MB-231 cells or MIA PaCa-2 (also known asPACA-2) cells were seeded at 1,000 cells per well in six-well plates andallowed to attach for 24 hours. MDA-MB-231 and MIA PaCa-2 cells werecultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS)and 5 ml Penicillin/Streptomycin at 37° C. in 5% CO₂. MDA-MB-231 and MIAPaCa-2 were established in estrogen receptor-negative human breastcancer and pancreatic carcinoma cell lines, respectively.(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewere each dissolved at a concentration of 10 mM in DMSO, and separatelyadded to cells at a concentration of 0.1, 0.25, 0.5, 1 or 2 μM. Controlplates received DMSO alone, at the same percentage of total culturevolume as that administered in conjunction with the highestconcentration of drug. Cell cultures were observed daily for 10-15 days,then fixed and stained with modified Wright-Giemsa stain (Sigma).Treatment with(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneresults in cell death of MDA-MB-231 cells or Paca-2 cells. See, e.g.,FIG. 2. The IC₅₀ for(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas found to be 0.5 μM. The IC₅₀ value for(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas found to be 0.5 μM.

Example 17

MDA-MB-231 cells (ATCC #HTB-26), grown in DMEM plus 15% heat inactivatedfetal bovine serum plus 10 mM HEPES pH 7.5, were plated in 60 mm² plates(2×10⁵ cells per plate). After two days candidate compounds in DMSO atvarious concentrations were diluted in media and added to individualplates such that the final DMSO concentration in the cell culture mediawas 0.1%. After two days incubation the culture was trypsinized, cellswere washed with media, counted using a hemocytometer and 500 cells,including cell bodies, were plated in 100 mm² plates in media. Two weekslater the media was removed and the cell colonies were fixed withmethanol for 10 minutes, stained with 1% crystal violet for 10 minutes,washed with water and air dried. Cell colonies were visually countedwhen there were greater then 50 cells present per colony. Platingefficiency was defined as the average number of colonies formed dividedby 500. The surviving fraction was defined as the plating efficiency ofa candidate compound divided by the plating efficiency of DMSOmultiplied by 100. For candidate compound titrations, the IC₅₀ value wasdetermined by fitting the equation y=Ae^(Bx) to the data points andextrapolating the concentration where surviving fraction equaled 50.Treatment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneresults in cell death of MDA-MB-231 cells. See, e.g., FIG. 3. The IC₅₀for(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas found to be 0.62 μM. The IC₅₀ for(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas found to be 4.1 μM.

Example 18

Recombinant Protein Kinase C (Calbiochem) (100 ng) was incubated with(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat 0.05, 0.5, or 10 μM for 15 minutes at room temperature. Subsequently,a radioactive labeling mix in kinase buffer (20 mM Tris-HCl pH 7.5, 10mM MgCl₂) containing 20 μM ATP, 0.2 μCi/μl γ³²P-ATP, 0.2 μg/μl HistoneH1 (Upstate Biotechnology/Millipore, Bedford, Mass.) was added to eachsample. The kinase reaction was carried out for 5 minutes at roomtemperature. Reaction products were analyzed by 12% SDS-PAGE andautoradiography.

Treatment of recombinant Protein Kinase C for 15 minutes at roomtemperature with(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat the tested concentrations did not reduce kinase activity incomparison to treatment with carrier alone. See, e.g., FIG. 4.

Example 19

MDA-MB-231 cells were serum-deprived overnight (16 hours) in the absenceor in the presence of the indicated concentrations of the separateenantiomers(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Cells were treated with 100 ng/ml recombinant human Hepatocyte GrowthFactor/Scatter Factor (HGF/SF) (R&D Systems #294-HG) for 10 minutes.Whole cell extracts were prepared in lysis buffer (20 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mMsodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 ng/mlleupeptin, 1 mM phenylmethylsulfonyl fluoride) and sonicated. Proteinconcentration was measured by Bradford assay using the BioRad reagent(BioRad, Hercules, Calif.), according to the manufacturer's directions.Samples (50 ng of protein) were resolved by 8% SDS-PAGE under reducingconditions and transferred onto a PVDF membrane (BioRad). The membranewas incubated 1 hour in TBS-T (50 mM Tris-HCl (pH=7.6), 200 mM NaCl,0.05% Tween 20) with 5% milk. Proteins were detected by incubationovernight at 4° C. in TBS-T with 5% milk and either a polyclonalantibody against phosphorylated c-Met (#3121) or a monoclonal antibodyagainst β-actin (A-5441) (Sigma), which was used as a control for totalprotein loading. After extensive washing in TBS-T, a horseradishperoxidase-conjugated anti-rabbit IgG (1:5000) or anti-mouse IgG(1:2000) (Amersham Biosciences) was added for 1 hour, and specificprotein bands were visualized using an enhanced chemiluminescencedetection system (Amersham Biosciences), according to the manufacturer'sinstructions. See, e.g., FIG. 5.

Treatment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinhibits both basal and HGF-induced autophosphorylation of c-Met at aconcentration of at least 30 nM. In contrast,(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehad only a minimum inhibitory effect on c-Met phosphorylation at muchgreater concentrations (20 μM). See, e.g., FIG. 5.

Example 20

A549 human lung cancer cells in a 96-well plate (Costar 3603,5,000/well) were treated with either A) DMSO as a control; B) 1.2 μM(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefor 38 hours before addition of 1:200 fluorescent Annexin V (green) and1:500 Propidium iodide (magenta, final concentration of 1 μg/mL). Thelabeling procedure was allowed to process at 37° C. for 20 minutesfollowed by image acquisition and analysis using an IC100 ImageCytometer (Beckman Coulter, Inc) with 10× amplification.

To determine whether(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneworks primarily through a cytostatic or apoptotic mechanism, cancercells exposed to(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewere stained with fluorescently labeled Annexin V (green fluorescence)and propidium iodide (bright magenta fluorescence). Annexin V is awell-validated reagent that specifically binds with high affinity toexternalized membrane phosphatidylserine, an early marker of the onsetof apoptosis, while propidium iodide is a marker for dead cells.Incubation of human lung cancer cells (A549) with 1.2 μM(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefor 38 hours induced cells to undergo apoptosis as evidenced by strongAnnexin V staining. A small percentage of cells (˜10-20%) co-stain withboth Annexin V and propidium iodide, indicating that a sub-population of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionetreated cells were already dead within 38 hours. These data areconsistent with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinducing cell death largely through activation of apoptotic mechanisms.(See, FIG. 6)

Example 21

MDA-MB-231 cells were pretreated with indicated concentrations of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefor 24 hours. 300 μl of each cell suspension (at a concentration 0.5×10⁶cells/mL in serum free medium) was placed in individual inserts andincubated for 24 hours at 37° C. The bottom wells housing the insertscontained 500 μl of 10% FBS containing medium. At 24 hours the mediumfrom each insert was aspirated, and cells that failed to invade weregently removed from the interior of the inserts with a cotton tippedswab. Each insert was then transferred to a clean well containing cellstain solution and incubated for 10 minutes at room temperature. Thebottom of the insert was destained by incubating in extraction solutionand OD was measured at 560 nM.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinhibited the migration across interstices in confluent cultures ofMDA-MB-231 cancer cells. The data represent the mean of two independentexperiments. (See FIG. 7)

The morbidity and mortality resulting from most cancers is the result oflocal invasion and metastasis from the primary tumors to other tissues.This process mostly depends on the motility and growth of tumor cells.Activation of c-Met by HGF induces a variety of cellular responsesincluding motility, invasion, wound healing and tissue regeneration. Ithas been established that aberrant activation of c-Met plays a criticalrole in the development and progression of primary tumors and secondarymetastases. HGF has the ability to dissociate epithelial sheets and tostimulate cell motility and invasion through extracellular matrixsubstrates, and HGF production correlates with tumor metastasis in vivo.

As shown above,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinhibited the invasive phenotype of MDA-MB-231 breast cancer cells withan estimated IC₅₀ value of approximately 500 nM. Similar results wereseen with brain and lung cancer cells. That is, the results show that(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinhibits metastatic cancer cell invasion.

Example 22

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneshows efficacy in a human breast cancer xenograft. MDA-MB-231 humanbreast cancer cells were inoculated subcutaneously into female athymicnude mice (8.0×10⁶ cells/mouse) and allowed to form palpable tumors.Once the tumors reached approximately 60 mm³, the animals were treatedorally with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat 200 mg/kg or vehicle control daily (5 consecutive days, followed by a2 day dosing holiday).(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas formulated in PEG 400:20% Vitamin E TPGS (60:40). The animalsreceived a total of 20 doses of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor vehicle control. Tumors were measured throughout treatment and thepost-treatment observation period. Each point represents the mean±SEM often tumors. (See, FIG. 8)

Treatment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneas monotherapy was effective at slowing tumor growth. Tumor growthinhibition of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas calculated to be 79% and was statistically significant (p=0.009).There was no significant change in body weight due to oraladministration of the vehicle or(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat 200 mg/kg.

Example 23

In a human colon cancer xenograft model HT-29 human colon cancer cellswere inoculated subcutaneously into female athymic nude mice (5×10⁶cells/mouse) and allowed to form palpable tumors. Once the tumorsreached approximately 60 mm³, the animals were treated orally witheither(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat 200 mg/kg or 300 mg/kg, or vehicle control daily (5 consecutive days,followed by a 2 day dosing holiday).(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas formulated in PEG 400:20% Vitamin E TPGS (60:40). The animalsreceived a total of 20 treatments of either(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneor vehicle control. Tumors were measured throughout treatment and thepost-treatment observation period. Each point represents the mean±SEM often tumors. (See, FIG. 9, Panel A)

In this highly aggressive colon xenograft model, animals dosed witheither 200 mg/kg or 300 mg/kg as a monotherapy showed significant tumorgrowth inhibition, with 300 mg/kg being more efficacious than 200 mg/kg.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedosed at 200 mg/kg showed an optimal tumor growth inhibition of 39%(p=0.006), while 300 mg/kg showed an optimal tumor growth inhibition of55% (p=0.00001). There was no significant change in body weight due tooral administration of either vehicle control or(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat 200 mg/kg or 300 mg/kg.

The efficacy of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas also tested in xenograft models with several other cancer celllines: human pancreatic cancer MIA PaCa2 (FIG. 9, Panel B), humanprostate cancer PC3 (FIG. 9, Panel C) and human gastric cancer MKN45(FIG. 9, Panel D). The cells were treated with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneas a monotherapy at indicated concentrations. In all these models,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionesignificantly inhibited tumor growth.

Example 24

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneshowed significant cytotoxicity against multiple cancer cell lines.Various cancer cell lines were treated with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat indicated concentrations ranging from 0.03 to 30 μM. The sensitivityof these cells to(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas measured by a standard cytoxicity MTS assay (FIG. 10). Human cancercell lines expressing c-Met and/or phospho-c-Met were sensitive to(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.In contrast, human cancer cell lines with no immunodetectable c-Met orphospho-c-Met (FIG. 10, SK-MEL-28, MCF-7 and NCI-H661) showed littlesensitivity.

Example 25

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewas assessed for its activity to inhibit a large panel (n=230) of humankinases. The data shown in Table 2 demonstrated that(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinhibited only c-Met to a significant degree and demonstrated modestactivity against a small number of other kinases.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneshowed an inhibitory constant (K_(i)) of ˜360 nM against c-Met.

TABLE 2 Kinase IC₅₀ CAMKIIδ ~10 μM Flt4 ~16 μM PAK3 ~6.6 μM  Pim-1 10 μM(33% inhibition)

Example 26

A human colon cancer xenograft model with HT-29 human colon cancer cellswas established as described in Example 23. The tumor-bearing mice weretreated with a single dose of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(300 mg/kg). The reduction of phospho-c-Met was detectedimmunohistochemically 24 hours later. A significant decrease in theamount of phospho-c-Met was visualized with the use of animmunoperoxidase system employing diaminobenzidine, which yields aninsoluble brown reaction product (FIG. 11, Panel A). Western blotting ofthe tumor sample for phospho-c-Met confirmed the immunohistochemicalanalysis (FIG. 11, Panel B).

Example 27

The present example describes the inhibition of c-Met Receptor TyrosineKinase in Clear Cell Sarcoma and MiT (Microphthalmia TranscriptionFactor)-Associated Tumors. Compounds of the invention also demonstratedefficacy in a patient with clear cell sarcoma. The studies described inthe present example used(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione,a small molecule inhibitor of the c-Met receptor tyrosine kinase.

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis a selective inhibitor of c-Met, a receptor tyrosine kinase. Whenabnormally activated, c-Met plays multiple roles in aspects of humancancer, including cancer cell growth, survival, angiogenesis, invasionand metastasis. The data described above demonstrate that(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneinhibits c-Met activation in a wide range of human tumor cell lines,including clear cell sarcoma, and show anti-tumor activity againstseveral human tumor xenografts. In clinical studies, treatment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionehas been well tolerated and has resulted in tumor responses andprolonged stable disease across broad ranges of tumors and doses.

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas administered to a patient with clear cell sarcoma. In particular,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionedemonstrated a partial response, as defined by RECIST (ResponseEvaluation Criteria in Solid Tumors), in a patient with clear cellsarcoma.

The objective response to(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneadministration was seen in a cohort of patients affected by amolecularly-linked group of sarcoma tumor types for which there is noeffective treatment. Based on this response,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis administered at a dose of 360 milligrams (mg) twice daily (b.i.d.).

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis administered to patients with MiT (Microphthalmia TranscriptionFactor)-associated tumors. MiT tumors, which can include clear cellsarcoma (CCS), alveolar soft part sarcoma (ASPS) andtranslocation-associated renal cell carcinoma (RCC), are linkedbiologically through a common chromosomal abnormality that isresponsible for the over-expression of c-Met resulting in thedevelopment of these tumors. Tumors with this abnormality are resistantto current therapies and, in the absence of successful surgicalresection, are invariably fatal.

During the first stage of a study, 23 patients were enrolled and treatedwith 120 mg of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneb.i.d. Fourteen of these patients were shown to be evaluable forefficacy. In addition to the patient with the confirmed partialresponse, ten of the evaluable patients have demonstrated stabledisease.

The objective clinical response demonstrated for(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionebuilds upon the data which showed that knockout of MiT expression byshRNA suppressed c-Met expression and impeded the growth of human clearcell sarcoma cells in vitro and in vivo. This finding led to thedevelopment of a clinical trial in patients with MiT-associated tumorsusing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione,which has shown anti-cancer activity, including objective tumorresponses, as well as the ability to inhibit the c-Met protein in tumorbiopsies from patients treated with the drug.

1. A polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8 and 14.1° 2θ using Cu Kα radiation.
 2. Thepolymorph of claim 1 characterized by an X-ray powder diffractionpattern comprising peaks at approximately 8.2, 10.8, 14.1, 15.5, 17.8,19.9 and 25.6° 2θ using Cu Kα radiation.
 3. The polymorph of claim 1characterized by an X-ray powder diffraction pattern comprising peaks atapproximately 8.2, 10.8, 14.1, 14.9, 15.5, 17.1, 17.8, 19.4, 19.9, 21.1,21.9, 23.0, 25.6 and 28.4° 2θ using Cu Kα radiation.
 4. A polymorph of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecharacterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9 and 12.0° 2θ using Cu Kα radiation.
 5. Thepolymorph of claim 4 characterized by an X-ray powder diffractionpattern comprising peaks at approximately 6.5, 9.9, 12.0, 16.7, 20.1 and22.8° 2θ using Cu Kα radiation.
 6. The polymorph of claim 4characterized by an X-ray powder diffraction pattern comprising peaks atapproximately 6.5, 9.9, 12.0, 13.2, 16.4, 16.7, 17.2, 20.1, 20.3, 20.8,22.8, 23.7, 28.6 and 30.4° 2θ using Cu Kα radiation.
 7. A(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane.
 8. A composition comprising(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane.
 9. The composition of claim 8 comprising greater than90%(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane.
 10. The composition of claim 9 comprising greater than95%(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane.
 11. The composition of claim 10 comprising greater than99%(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane.
 12. A(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.13. A composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.14. The composition of claim 13 comprising greater than 90%(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.15. The composition of claim 14 comprising greater than 95%(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.16. The composition of claim 15 comprising greater than 99%(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine.17. The composition of claim 13 comprising less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine.
 18. The composition of claim 17 comprising less than0.5%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine.
 19. The composition of claim 18 comprising less than0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionepseudoephedrine.
 20. A chirally purified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprising less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.21. The chirally pure(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneof claim 20 comprising less than 0.7%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.22. The chirally pure(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneof claim 21 comprising less than 0.5%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.23. The chirally pure(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneof claim 22 comprising less than 0.1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.24. A method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: a. mixing(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewith (1S,2S)-(+)-pseudoephedrine in a first solvent to form solid(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrine;b. washing the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinesolid formed in step (a) with an aqueous mixture of the first solvent;c. reacting the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrinefrom step (b) with an acid in an organic solvent and isolating theorganic layer of the resultant solution; d. washing the organic layerfrom step (c); e. adding a second solvent to the organic layer; f.concentrating the organic layer until the amount of the second solventin the solution is less than 5%; and g. crystallizing from the organiclayer in step (f) and drying the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionesolution under vacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.25. The method of claim 24, wherein said first solvent is a non-aqueoussolvent.
 26. The method of claim 25, wherein said non-aqueous solvent ismethanol, ethanol, acetonitrile, or a mixture thereof.
 27. The method ofclaim 24, wherein said second solvent is a non-aqueous solvent.
 28. Themethod of claim 27, wherein said non-aqueous solvent is methanol,ethanol, acetonitrile, or a mixture thereof.
 29. The method of claim 24,wherein said second solvent is the same as said first solvent.
 30. Themethod of claim 24, wherein said second solvent is different from saidfirst solvent.
 31. The method of claim 24, wherein said organic solventin step c is methyltetrahydrofuran
 32. The method of claim 24, whereinsaid organic layer is washed with a salt solution in step d.
 33. Themethod of claim 32, wherein said salt solution is a sodium chloridesolution.
 34. The method of claim 24, further comprising rinsing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal after step (g).
 35. The method of claim 34, wherein(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal is rinsed with an alcohol selected from ethanol and methanol.36. The method of claim 24, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.37. The method of claim 36, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 0.7%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.38. A method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: a. mixing(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewith cyclohexylethylamine in a first solvent to form solid(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.cyclohexylethylamine;b. washing the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.cyclohexylethylaminesolid formed in step (a) with an aqueous mixture of the first solvent;c. reacting the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.cyclohexylethylaminefrom step (b) with an acid in an organic solvent and isolating theorganic layer of the resultant solution; d. washing the organic layerfrom step (c); e. adding a second solvent to the organic layer; f.concentrating the organic layer until the amount of the second solventin the solution is less than 5%; and g. crystallizing from the organiclayer in step (f) and drying the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionesolution under vacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.39. The method of claim 38, wherein said first solvent is a non-aqueoussolvent.
 40. The method of claim 39, wherein said non-aqueous solvent isacetonitrile.
 41. The method of claim 38, wherein said second solvent isa non-aqueous solvent.
 42. The method of claim 41, wherein saidnon-aqueous solvent is methanol, ethanol, acetonitrile, or a mixturethereof.
 43. The method of claim 38, wherein said second solvent is thesame as said first solvent.
 44. The method of claim 38, wherein saidsecond solvent is different from said first solvent.
 45. The method ofclaim 38, wherein said organic solvent in step c ismethyltetrahydrofuran
 46. The method of claim 38, wherein said organiclayer is washed with a salt solution in step d.
 47. The method of claim46, wherein said salt solution is a sodium chloride solution.
 48. Themethod of claim 38, further comprising rinsing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal after step (g).
 49. The method of claim 48, wherein(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecrystal is rinsed with an alcohol selected from ethanol and methanol.50. The method of claim 38, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.51. The method of claim 50, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 0.7%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.52. A method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: a. mixing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.(1S,2S)-(+)-pseudoephedrineand an acid; b. adding an alcohol to the mixture from (a) to form aslurry; c. heating and stirring the slurry formed in (b); d. cooling andisolating(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione;e. washing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneisolated in step (d) with a first solvent; f. dissolving(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom step (e) in a second solvent to form a solution; g. adding a thirdsolvent to the solution in (f) and distilling the solution until theamount of said second solvent in the solution is less than 5%; h.crystallizing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom the solution in (g); i. optionally, adding a fourth solvent tomature the crystals from (h); j. isolating the crystals from (i) byfiltration; k. washing the crystals from (j) with a mixture of the thirdsolvent and fourth solvent; and l. drying the crystals from (k) undervacuum, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.53. The method of claim 52, wherein said alcohol is methanol, ethanol,or a mixture thereof.
 54. The method of claim 52, wherein said firstsolvent is a non-aqueous solvent.
 55. The method of claim 54, whereinsaid non-aqueous solvent is methanol, ethanol, or a mixture thereof. 56.The method of claim 52, wherein said second solvent is a non-aqueoussolvent.
 57. The method of claim 56, wherein said non-aqueous solvent istetrahydrofuran.
 58. The method of claim 52, wherein said third solventis a non-aqueous solvent.
 59. The method of claim 58, wherein saidnon-aqueous solvent is methanol, ethanol, or a mixture thereof.
 60. Themethod of claim 52, wherein said fourth solvent is an aqueous solvent.61. The method of claim 60, wherein said aqueous solvent is water. 62.The method of claim 52, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.63. The method of claim 62, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 0.7%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.64. A method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising: a. dissolving(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein dichloromethane and isolating the(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane; b. dissolving(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionedichloromethane in a first solvent; c. distilling the solution of step(b) until the level of dichloromethane in the solution is <0.1% byweight; d. diluting the solution of step (c) in a second solvent; e.introducing the solution of step (d) into a multicolumn chromatographysystem containing a packing suitable for chiral separation; f. poolingthe resultant raffinate obtained from the system in step (e); and g.crystallizing the raffinate from step (f) and filtering the resultant(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.65. The method of claim 64, wherein said first solvent is a non-aqueoussolvent.
 66. The method of claim 65, wherein said non-aqueous solvent ismethanol, ethanol, or a mixture thereof.
 67. The method of claim 64,wherein said second solvent is a non-aqueous solvent.
 68. The method ofclaim 67, wherein said non-aqueous solvent is methanol, ethanol,acetonitrile, or a mixture thereof.
 69. The method of claim 68, whereinsaid non-aqueous solvent is a mixture of methanol and acetonitrile. 70.The method of claim 64, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 1%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.71. The method of claim 70, wherein said produced(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises less than 0.7%(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.72. A method for preparing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,comprising steps a-f of claim 64 and step g′, wherein step g′ comprisesevaporating the raffinate in step f, thereby producing(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.73. A pharmaceutical composition comprising a polymorph of any one ofclaims 1-3 and 4-6 and a pharmaceutically acceptable carrier orexcipient.
 74. A pharmaceutical composition comprising a chirallypurified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneof any one of claims 20-23.
 75. A method of treating a cancer,comprising administering to a subject a therapeutically effective amountof a polymorph of any one of claims 1-3 and 4-6 in combination with apharmaceutically acceptable carrier.
 76. The method of claim 75, whereinsaid cancer is selected from lung cancer, colon cancer, breast cancer,pancreatic cancer, prostate cancer, chronic myelogenous leukemia,melanoma, ovarian cancer, renal carcinoma, hepatoma, brain cancer andmultiple myeloma.
 77. A method of treating a cancer, comprisingadministering to a subject a therapeutically effective amount of achirally purified(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneof any one of claims 20-23 in combination with a pharmaceuticallyacceptable carrier.
 78. The method of claim 77, wherein said cancer isselected from lung cancer, colon cancer, breast cancer, pancreaticcancer, prostate cancer, chronic myelogenous leukemia, melanoma, ovariancancer, renal carcinoma, hepatoma, brain cancer and multiple myeloma.